Abstract: In order to explore the function and mechanism of 2,4-epibrassinolide (EBR) in strengthening the ability of tobacco seedlings to resist drought, drought resistant cultivar Yuyan12 and drought sensitive cultivar Yuyan10 were grown under drought stress, and the effects of 2,4-epibrassinolide on growth of tobacco seedlings under drought stress were investigated by hydroponic experiments. The results showed that under drought stress, the growth of seedlings of Yuyan 12 and Yuyan 10 was inhibited, the O₂^- production rate and MDA contents in leaves of tobacco seedlings were increased, the Chlorophyll was destroyed, and the Photosynthetic rate was reduced. Under stress conditions, foliar spraying of EBR could significantly promote the growth and root development, effectively enhance the activities of antioxidase, and decrease the contents of superoxide anion, hydrogen peroxide and MDA. Meanwhile, the content of chlorophyll and the photosynthetic rate were increased. Compared with Yuyan12, drought stress had a greater influence on Yunyan10, but exogenous EBR could reduce the damage caused by drought on both varieties. Spraying exogenous EBR could promote the morphological development of root system, improve the activities of antioxidant enzymes, reduce the levels of reactive oxygen accumulation and membrane peroxidation, improve the photosynthetic ability, and enhance the drought resistance of tobacco.